Compositions and methods for reducing spliceopathy and treating rna dominance disorders

Abstract

The disclosure features compositions and methods for the treatment of disorders associated with improper ribonucleic acid (RNA) splicing, including disorders characterized by nuclear retention of RNA transcripts containing aberrantly expanded repeat regions that bind and sequester splicing factor proteins. Disclosed herein are interfering RNA constructs that suppress the expression of RNA transcripts containing expanded repeat regions, as well as viral vectors, such as adeno-associated viral vectors, encoding such interfering RNA molecules. For example, the disclosure features interfering RNA molecules, such as siRNA, miRNA, and shRNA constructs, that anneal to dystrophia myotonica protein kinase (DMPK) RNA transcripts and attenuate the expression of DMPK RNA containing expanded CUG trinucleotide repeats. Using the compositions and methods described herein, a patient having an RNA dominance disorder, such as a human patient having myotonic dystrophy, among other conditions described herein, may be administered an interfering RNA construct or vector containing the same so as to reduce the occurrence of spliceopathy in the patient, thereby treating an underlying etiology of the disease.

What is claimed is: 
     
         1 . A viral vector comprising one or more transgenes, each encoding an interfering ribonucleic acid (RNA) of at least 17 nucleotides in length, wherein each interfering RNA comprises a portion that anneals to an endogenous RNA transcript comprising an expanded repeat region, and wherein the portion of each interfering RNA anneals to a segment of the endogenous RNA transcript that does not overlap with the expanded repeat region. 
     
     
         2 . The viral vector of  claim 1 , wherein the endogenous RNA transcript encodes human dystrophia myotonica protein kinase (DMPK). 
     
     
         3 . The viral vector of  claim 2 , wherein the expanded repeat region comprises 50 or more CUG trinucleotide repeats. 
     
     
         4 . (canceled) 
     
     
         5 . The viral vector of  claim 2 , wherein the vector further comprises a transgene encoding a human DMPK RNA transcript that does not anneal to the interfering RNA. 
     
     
         6 . The viral vector of  claim 5 , wherein the human DMPK RNA transcript is less than 85% complementary to the interfering RNA. 
     
     
         7 . The viral vector of  claim 2 , wherein the endogenous RNA transcript comprises a portion having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         8 - 11 . (canceled) 
     
     
         12 . The viral vector of  claim 2 , wherein the portion of each interfering RNA has a nucleic acid sequence that is least 85% complementary to the nucleic acid sequence of a segment within any one of exons 1-15 of human DMPK, any one of introns 1-14 of human DMPK, an exon-intron boundary within human DMPK, or within the 5′ untranslated region (UTR) or 3′ UTR of human DMPK. 
     
     
         13 - 33 . (canceled) 
     
     
         34 . The viral vector of  claim 2 , wherein the portion of each interfering RNA anneals to a segment of the endogenous RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 3-39. 
     
     
         35 - 38 . (canceled) 
     
     
         39 . The viral vector of  claim 2 , wherein the interfering RNA comprises a portion having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-161. 
     
     
         40 - 42 . (canceled) 
     
     
         43 . The viral vector of  claim 1 , wherein the endogenous RNA transcript comprises human chromosome 9 open reading frame 72 (C9ORF72) and an expanded repeat region. 
     
     
         44 - 49 . (canceled) 
     
     
         50 . The viral vector of  claim 43 , wherein the portion of each interfering RNA has a nucleic acid sequence that is at least 85% complementary to the nucleic acid sequence of a segment within human C9ORF72. 
     
     
         51 - 60 . (canceled) 
     
     
         61 . The viral vector of  claim 1 , wherein the interfering RNA is a short interfering RNA (siRNA), a short hairpin RNA (shRNA), or a micro RNA (miRNA). 
     
     
         62 - 64 . (canceled) 
     
     
         65 . The viral vector of  claim 1 , wherein the interfering RNA is operably linked to a promoter that induces expression of the interfering RNA in a muscle cell or neuron. 
     
     
         66 . The viral vector of  claim 65 , wherein the promoter is a desmin promoter, a phosphoglycerate kinase (PGK) promoter, a muscle creatine kinase promoter, a myosin light chain promoter, a myosin heavy chain promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family promoter, an actin alpha promoter, an actin beta promoter, an actin gamma promoter, or a promoter within intron 1 of ocular paired like homeodomain 3 (PITX3). 
     
     
         67 . (canceled) 
     
     
         68 . The viral vector of  claim 1 , wherein the viral vector is an adeno-associated virus (AAV), optionally wherein the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, or AAVrh74 serotype. 
     
     
         69 . (canceled) 
     
     
         70 . The viral vector of  claim 68 , wherein the viral vector is AAV2/8 or AAV2/9. 
     
     
         71 - 74 . (canceled) 
     
     
         75 . A nucleic acid encoding or comprising an interfering RNA, wherein the interfering RNA comprises a portion having at least 85% sequence identity to the nucleic acid sequence of any one of SEQ ID NOs: 3-161. 
     
     
         76 - 116 . (canceled) 
     
     
         117 . A method of reducing the occurrence of spliceopathy in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the vector of  claim 1 . 
     
     
         118 . The method of  claim 117 , wherein the patient has myotonic dystrophy or amyotrophic lateral sclerosis. 
     
     
         119 - 124 . (canceled) 
     
     
         125 . A method of treating a disorder characterized by nuclear retention of RNA comprising an expanded repeat region in a human patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of the vector of  claim 1 . 
     
     
         126 - 130 . (canceled)